PROJECT SUMMARY Antimicrobial resistance, especially in Gram-negative bacterial pathogens, constitutes an urgent threat to human health. The discovery and development of new chemical matter inhibiting essential bacterial targets constitutes one powerful approach to addressing this issue. Novel Bacterial Type II Topoisomerase Inhibitors (NBTIs) derive their antibacterial activity from inhibition of the clinically validated enzymes DNA gyrase and topoisomerase IV (TopoIV). A novel binding mode that obviates target-based cross-resistance to fluoroquinolones has established NBTIs as a new class of antibiotics. However, the ultimate potential of the NBTI class to treat infections caused by Gram-negative pathogens has been limited by cardiovascular safety concerns as well as by the intrinsic challenges of achieving high intracellular concentrations in Gram-negative bacteria. This proposal elaborates strategies to address these key issues that have limited the advancement of the field. We have previously synthesized innovative dioxane-linked NBTIs directed at Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus. Broad spectrum antibacterial screening revealed that representative compounds also possess promising activity against Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii. These rationally-designed inhibitors also demonstrate attenuated hERG inhibition as compared to structure-matched controls, offering the promise of improved cardiovascular safety. In this proposal, we hypothesize that the incorporation of specific molecular features, such as a primary amine and reduced conformational flexibility, will enable further improvements in Gram-negative antibacterial activity while preserving a favorable safety profile. We will determine minimum inhibitory concentrations (MICs) and enzymatic (gyrase and TopoIV) IC50 values from a panel of relevant Gram-negative bacteria (P. aeruginosa, A. baumannii, and E. coli). We will utilize our synthetic expertise to interrogate this hypothesis using multiple chemical series, one involving direct modification of our earlier NBTIs and one targeting a restructured NBTI pharmacophore. Our interdisciplinary team will rigorously test these hypotheses according to the following specific aims: 1) Optimize dioxane-linked NBTIs for Gram-negative activity a. Incorporate a primary amine b. Reduce conformational flexibility 2) Redesign NBTI enzyme-binding moiety to incorporate a primary amine 3) Quantify potency with enzymes and cells; measure hERG inhibition Successfully achieving our aims will deliver NBTI lead molecules with enhanced safety and whole cell antibacterial activity against critically important Gram-negative pathogens.